Transient Recombinant Protein Expression in Mammalian Cells

Transient gene expression has evolved into an attractive technology for the rapid production of milligram to gram amounts of recombinant proteins. This review describes the different methods for introducing foreign DNA into suitable mammalian cells with either viral or non-viral vectors. Particular emphasis is given to non-viral transient transfection which represents meanwhile the most prominent variant due to recent progress in the resulting protein productivity. Non-viral transient transfection protocols are always based on the use of specific transfection reagents or the application of an electroporation device. The corresponding methods are compared with regard to their scale-up potential, also in consideration of potential production costs. The underlying cellular pathways of plasmid DNA incorporation, cytoplasmic release and translocation into the nucleus are important details to understand the transfection principle and further improve the technology. Problems associated with the application of transient gene expression at a larger scale are also addressed. In particular, the requirement of different cell culture media conditions for plasmid DNA complex preparation (if necessary), the transfection process itself and a high titer recombinant production need to be harmonized. Strategies to improve recombinant protein productivity by increasing the cell-specific output and/or sustaining the production phase are itemized as well. This can be accomplished by enabling cells to perform episomal plasmid replication, co-transfection with other plasmids, altering the cellular metabolism, temperature reduction, supplementation of specific production enhancers or combinations thereof. A number of examples for successful applications at pilot scale are also provided.